A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli

Abstract

Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.

Ammonia Production Using Bacteria and Yeast toward a Sustainable Society

“…This nitrogenase uses molybdenum as a cofactor, but Azotobacter vinelandii exists as a nitrogen-fixing bacterium with a nitrogenase that uses vanadium and iron. Various attempts have already been made with some success to construct heterologous expression of this nitrogenase gene cluster in E. coli and yeast, to elucidate the mechanism by which it is protected from oxygen, and to increase its activity [81][82][83]. For example, heterologous expression of the nitrogenase complex of A. vinelandii has been successful in E. coli, and the minimum number of genes required for nitrogen fixation has been elucidated.…”

Trends in Biological Ammonia Production

“…Although the reaction mechanism of nitrogenase is unclear due to its interrelated multiple subunits, scientists have attempted to construct a heterologous expression system for Klebsiella nitrogenase subunits in E. coli [37]. Similarly, an heterologous expression of the Klebsiella nitrogenase gene cluster has been constructed in E. coli and yeast to understand the mechanism by which nitrogenase functions without oxygen as well as to increase its activity [38,39]. Various studies have also investigated how nitrogen-fixing bacteria can function under aerobic conditions without inactivating nitrogenase.…”

“…Various studies have also investigated how nitrogen-fixing bacteria can function under aerobic conditions without inactivating nitrogenase. Such researches involve the use of polysaccharide membrane to protect nitrogenase from oxygen exposure [39,40]. For industrial applications of nitrogen-fixing bacteria, companies such as Pivot Bio, Inc. and Ginkgo Bioworks engineered Enterobacter sp.…”

Trends in Biological Ammonia Production

“…Although the reaction mechanism of nitrogenase is unclear due to its multiple interrelated subunits, scientists have attempted to construct a heterologous expression system for Klebsiella nitrogenase subunits in E. coli [ 45 ]. Similarly, heterologous expression of the Klebsiella nitrogenase gene cluster has been constructed in E. coli and yeast to understand the mechanism by which nitrogenase functions without oxygen as well as to increase its activity [ 46 , 47 ]. Various studies have also investigated how nitrogen-fixing bacteria can function under aerobic conditions without inactivating nitrogenase.…”

“…Various studies have also investigated how nitrogen-fixing bacteria can function under aerobic conditions without inactivating nitrogenase. Such research involves the use of polysaccharide membranes to protect nitrogenase from oxygen exposure [ 47 , 48 ].…”